Composition for delivering DNA into antigen presenting cells

ABSTRACT

A molecular delivery complex specific to antigen-presenting cells is formed from a a non-viral gene delivery system complexed with foreign genetic material. The complex then enters the targeted cells through a specific receptor and overcomes the degradation mechanism, so that functional uptake of the foreign genetic material, or transduction, of the cell, results in gene expression. The invention also includes a method for genetic immunization without a needle.

This application is a continuation-in-part of U.S. Ser. No. 60/058,933,filed Sep. 15, 1997, which is incorporated herein as if set forth infull.

FIELD OF THE INVENTION

The present invention relates generally to methods and compositions fordelivering foreign genetic material into cells. Specifically, it relatesto a technique for receptor-mediated delivery of genes to cells. A genedelivery complex compatible with a specific type of targeted cell isformed from the foreign genetic material, a vector, and optionally, acarrier. The complex is then exposed to the cells under conditionspermitting receptor-mediated endocytosis, resulting in the functionaluptake, or transduction, of the foreign genetic material. The method isnot only useful for in vitro, but also in vivo gene delivery to antigenpresenting cells, specifically described as transcutaneous gene transferto skin Langerhans cells. This technique is particularly useful forpreventive and therapeutic genetic immunization when the foreign geneticmaterial is an immunogen such as DNA encoding a substantial portion ofthe antigens and particles associated with an infectious virus, andwhere delivery by injection is undesirable.

BACKGROUND OF THE INVENTION

The immune system for animals has two different but related responses,the cellular immune response and the humoral immune response. Thecellular immune response produces T lymphocytes which kill cells havingforeign identifying markers on their surface. Cells which have suchidentifying markers on their surface are said to “present” an antigen,and are referred to as antigen presenting cells (APCs). In addition, Tlymphocytes also stimulate the humoral response by helping B cells, theprecursors of plasma cells.

The humoral immune response results in the production by plasma cells ofantibodies which act on specific molecules in solution. Antibodies (orimmunoglobulins) are proteins synthesized by an animal in response tothe presence of a foreign substance. They are secreted by plasma cells,which are derived from B lymphocytes (B cells). These soluble proteinsare the recognition elements of the humoral immune response. Eachantibody has specific affinity for the foreign substance that stimulatedits synthesis. That is, the antibody has a segment or site, called anantigen binding site, which will adhere to the foreign substance. Aforeign macromolecule capable of eliciting the formation of antibodiesagainst itself is called an antigen. Proteins and polysaccharides areusually effective antigens. The specific affinity of an antibody is notfor the entire macromolecular antigen, but for a particular site on itcalled the antigenic determinant or epitope. Antibodies recognizeforeign molecules in solution and on membranes irrespective of themolecule's context. The humoral immune response is most effective incombating bacteria and viruses in extracellular media. (The word humoris the Latin word for fluid or liquid.) One strategy for conferringimmunity against disease is to expose the individual to one or moreantigens associated with a virus or bacterium rather than use the actualvirus or bacterium. Such a vaccine is known as a subunit vaccine, and itworks particularly well to stimulate the production of antibodies.

T cells mediate the cellular immune response. In contrast to the humoralimmune response, the cellular immune response destroys virus-infectedcells, parasites, and cancer cells. The surface of T cells containtransmembrane proteins called T cell receptors that recognize foreignmolecules on the surface of other cells. That is, T cells recognizeantigen presenting cells (APCs). T cell receptors do not recognizeisolated foreign molecules. The foreign unit must be located on thesurface of a cell, and must be presented to the T cell by a particularmembrane protein, one encoded by a highly variable chromosomal region ofthe host known as the major histocompatibility complex (MHC). The MHCencodes three classes of transmembrane proteins. MHC Class I proteins,which are expressed in nearly all types of cells, present foreignepitopes to cytotoxic T cells. MHC Class II proteins, which areexpressed in immune system cells and phagocytes, present foreignepitopes to helper T cells. MHC Class III proteins are components of theprocess know as the complement cascade.

There are a variety of T cells, including cytoxic T lymphocytes (CTL, orkiller T cells) which destroy cells which display a foreign epitopebound to an MHC protein. When the foreign-epitope-plus-MHC-protein bindsto the T cell receptor, the T cell secretes granules containingperforin, which polymerizes to form transmembrane pores, therebybreaking the cell open, or inducing cell lysis. Other classes of Tcells, called Helper T cells, secrete peptides and proteins calledlymphokines. These hormone-like molecules direct the movements andactivities of other cells. Some examples are Interleukin-2 (IL-2),Interleukin-4 (IL-4), Interferons, Granulocyte-Macrophagecolony-stimulating factor (GM-CSF), and Tumor necrosis Factor (TNF). TheT cells are implicated in the complement cascade, a precisely regulated,complex series of events which results in the destruction ofmicroorganisms and infected cells. More than fifteen soluble proteinsco-operate to form multi-unit antigen-antibody complexes that precedethe formation of large holes in the cells' plasma membrane.

Expression of foreign genes in antigen presenting cells (APC) may beused to generate efficient CTL response in animals. Therefore, genetransfer and genetic modification of APC has potential to generateeffective vaccine and therapeutic approaches against different diseases,including viral infections and cancer. Live recombinant virus vectorsexpressing various foreign antigens, such as pox viruses, adenoviruses,and retroviruses, can be used to elicit both humoral and cellular immuneresponse by mimicking viral infection. Also, live attenuated (or,weakened) viruses have been proposed as vaccines. DNA vaccinationstrategy is also being explored. Different viral genes have been clonedinto plasmid DNA and injected into muscles, skin, or subcutaneously.These constructs are able to express proteins and elicit both a cellularand humoral immune response.

It has been suggested that viral diseases may be responsive to thetechnique of genetic immunization. Certain cells, such as dendriticcells, are known to pick up antigens and migrate from the tissues of thebody to the lymphoid tissues. There these cells present the antigens inthe lymphoid organs: that is, they display a foreign epitope bound to anMHC protein. Such antigen-presenting cells (APCs) are a known part ofthe immune response mechanism. If cells such as a dendritic cells (DC)are modified so that they contain DNA encoding a virus which isinfectious but incapable of efficient reproduction, they could not onlypresent antigens in the classic sense, but also be manipulated toproduce, or express, viral particles and a wide variety of viralproteins. A novel technology has been described in U.S. Ser. No.08/803,484 “Methods and Compositions for Protective and TherapeuticGenetic Immunization” which is incorporated herein by reference as ifset forth in full. It discloses that genes of a replication-incompetentvirus can be incorporated into antigen presenting cells which thenmigrate to the lymphoid organs and produce the full complement of viralantigens and viral particles, thereby triggering both humoral andcellular immune responses. It teaches that DC in the lymphoid organs maythen express all viral antigens and produce “authentic looking” viralparticles. These viral particles would therefore play a pivotal role inthe generation of additional immune responses.

This reference describes in Example 13 “in vivo transduction” of cellsincluding APC. In that example, several well known methods includingviral and non-viral gene delivery are exemplified. In Example 14 “invivo transduction” of cells including APC are described. These utilize(1) direct DNA injection; (2) injection of liposomes or virosomescontaining the DNA; (3) direct intersplenic injection of Class 4 poxviruses; and (4) rectal and vaginal suppositories carrying gene deliveryvehicles. However, this reference did not describe in detail the methodsof in vitro and in vivo gene delivery. That is the subject of thepresent invention.

There is some evidence suggesting that genetic modification of APC willbe effective to vaccinate both neonatal and adult animals and humans.Ridge et al. (Science 271: 1723-1726, 1996) have injected DC expressinga foreign antigen isolated from another animal intravenously into mice.Both neonatal and adult mice injected with these DC were able togenerate good CTL killing of target cells. These experiments alsodemonstrated that DC expressing a foreign antigen can induce protectivecell-mediated immune response which is able to eliminate infected cellsin case of viral infections. In addition, these experiments demonstratedthat DC-mediated immunization of neonates may be possible. Theseexperiments did not use genetically modified cells, nor did they utilizeseveral foreign antigens nor a virus as described in the presentinvention.

Sarzotti et al. (Science 271: 1726-128, 1996) demonstrated that low doseinoculation with viruses results in a protective immune response(Th1-type) which generates CTL response but high dose inoculation willresult in a nonprotective (Th2-type) immune response which mainlygenerates antibodies. These CTL responses were very long lasting andalso could be generated in neonates. High doses of virus might overwhelmand disarm T-cells before DC could activate the T-cells. Again, theroute of administration, not through injection but through presentationby DC, is important. These findings are consistent with other resultsshowing that exposure to low dose viruses provokes predominantlycellular (Th1-type) immune response. In macaques, a low dose SIV primedthe Th1-type response without antibody production and protected animalsagainst high dose challenge (Clerici et al. AIDS 8: 1391-1395, 1994). Inhumans, similar results were demonstrated by Rowland-Jones et al.(Nature Med 1: 59-64, 1995)

The process of modification of cells so that they contain foreigngenetic material is called gene transfer, transfection or transduction.None of the papers cited herein have presented evidence of efficientgene transfer to antigen presenting cells, either in vitro or in vivo.As background for gene transfer into antigen presenting cells such asDC, several “low efficient” in vitro methods have been described,including liposome-mediated gene transfer; electroporation andretrovirus-vector- and adenovirus-vector-mediated gene transfers(Arthur, J. F et al. Cancer Gene Therapy. 4:1 17-21, 1997, Song, E. S.et al. Proc Natl Acad Sci U S A 94:5, 1943-8, 1997). All of these invitro methods involve the isolation of large populations of cells whichare treated in the laboratory with a gene delivery vehicle. All human oranimal applications involve the reintroduction of these geneticallymodified cells. Therefore, in vitro gene delivery methods are notfeasible for vaccination or treatment of large numbers of individuals.Known in vivo methods include intradermal or intramuscular injection ofrecombinant virus vectors and intradermal, subcutaneous andintramuscular injection of plasmid DNA. None of these methods have beenshown to effectively deliver genes into antigen presenting cells, suchas dendritic cells, much less delivery of genes through the skin intothe Langerhans cells.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1 illustrates antibody mediated gene delivery into cells expressingFc-receptors.

FIG. 2 illustrates gene delivery into dendritic cells and Langerhanscells via the mannose-receptor using PEI-man-DNA complex

FIG. 3 illustrates the transcutaneous gene delivery approach

FIG. 4 compares effectiveness of in vitro transfection of human DC usingtwo different complexes of the present invention.

FIG. 5 CTL assay, illustrates that transduced DC are able to generatecytotoxic T cells from naive T cells: compares % cytotoxicity of DCtransfected with integrase-HIV plasmid to that of control DC.

FIG. 6 illustrates the CTL response after ex vivo genetic immunizationcompares CTL response obtained in vivo using transfected DC.

FIGS. 7A-7D is a color photograph showing that transcutaneous geneticimmunization results in gene expression in the lymphoid organs.

DETAILED DESCRIPTION OF THE DRAWINGS

In FIG. 1, the process for antibody mediated gene delivery into cellsexpressing Fc-receptors is illustrated conceptually. Target cells (1)having one or more receptors (2 a,b,c,d) are exposed to a gene-deliverycomplex (3) comprising a carrier (4) and a vector (5) which includes theforeign genetic material. The gene delivery complex (3) binds to thereceptors (2 a,b,c,d) of the cell (1) and the vector (4) is incorporatedinto the cell via endocytosis or phagocytosis in an endosome (6). Thevector (4) has the property of breaking the endosome (6), allowing theforeign genetic material to be released into the cell.

In FIG. 2, the process for sugar-mediated gene delivery into cellsexpressing mannose-receptors is illustrated conceptually. Target cells(10), in this case, immature Langerhans cells having one or moremannose-receptors (12) are exposed to a gene-delivery complex (13)comprising a polyethylenimine-sugar (mannose) complexed with the foreigngenetic material. The gene delivery complex (13) binds to the receptors(12) of the cell (10) and the PEI-man-DNA is incorporated into the cellvia endocytosis in an endosome (14). The vector (PEI-man) has theproperty of breaking (15) the endosome, allowing the foreign geneticmaterial to be released into the cell. The cell matures (16) andexpresses proteins (17) coded by the foreign genetic material.

In FIG. 3, the experiment demonstrating in vivo the sugar-mediated genedelivery into cells expressing mannose-receptors is illustrated. Targetcells are Langerhans cells in the skin known to express mannosereceptors. Mice (21) were anesthetized and an area on the back of eachmouse (22) was shaved. The shaved surface with ethanol. PEI-man-DNA genedelivery complex in 8% glucose (23) was applied to the shaved area (22)of each mouse. Langerhans cells (24) found in the shaved area of theskin (22) pick up the complex as described in FIG. 2 above, getactivated and migrate (24) to the draining lymph node (25). During themigration Langerhans cells (24) mature to be dendritic cells (26) andexpress the protein (27) encoded by the DNA.

In FIG. 4, experimental results are demonstrating the in vitro genedelivery with PEI-DNA vs. PEI-man-DNA complexes. Human DC were culturedas described in the text and transfected with complexes. A marker geneencoding a green fluorescent protein (GFP) was used as the DNA. In theseexperiments the complexes were dissolved in a solution of NaCl. Theexperiment demonstrated that PEI-man is more efficient to transfectcultured DC than PEI.

FIGS. 5-6 report experimental results and are discussed in detail in theExample section, below.

FIGS. 7A-7D reports experimental evidence that transcutaneoustransduction of Langerhans cells results in migration of the cells andexpression of the transferred gene. The figure is a series of colorphotographs which records green cells having DC morphology andexpressing the green fluorescent protein, which is the product of thegene which was transferred via skin delivery. Panel A is a sample of alymph node from a control mouse at 200× magnification. It exhibits anormal amount of background fluorescence. The same is true of Panel C,except that the magnification is 400×. Panel B is a sample from a lymphnode of a mouse that was immunized by the transcutaneous application ofa PEI-mannose-DNA complex. Panel D is the same as Panel B, except thatthe magnification is 400×. The fluorescence exhibits the bumpymorphology characteristic of dendritic cells expressing proteins.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method of improvedefficiency of gene transfer to cells, in vitro and in vivo. A furtherobject of this invention is to provide an improved method of geneticimmunization by increasing the efficiency of gene transfer to antigenpresenting cells. It is yet a further object of this invention toprovide a means of stimulating both humoral and cellular immuneresponses to the protein product of the transferred genetic material.Yet another object of this invention is to provide an effective immuneresponse to viral diseases. Yet another object of this invention is toprovide a vaccine for viral diseases which is effective and has improvedsafety. An advantage of the present invention is that it provides a invivo gene transfer method which can be utilized for immunotherapy andvaccination for a wide variety of diseases.

An another advantage of the present invention is that it can utilize anytype of DNA, or RNA, including plasmid DNA encoding immunogens likeoncogens, immunogens (causing allergy), viral proteins or differenttypes of replication defective viruses, defective viral particles, aswell as plasmid DNA.

An another advantage of the present invention is that it can utilizeinstead of DNA proteins like oncogenic protein (e.g. mutated p53 orRas), immunogens (causing allergy), viral proteins or different types ofreplication defective viruses.

These and other objects and advantages of the present invention willbecome apparent through the text and examples herein.

The objects and advantages of the present invention are achieved byforming a gene delivery complex comprising a vector (which contains thedesired foreign genetic material) and a carrier (which can bind both tothe cells and to the gene delivery particle), then exposing target cellsto the complex under conditions permitting endocytosis. The vector hasthe characteristic that it allows the genetic material to escape fromendosomal degradation and it delivers the desired foreign geneticmaterial to either the cytoplasm or to the nucleus. Foreign proteins canbe expressed and presented to the immune system by the geneticallymodified cells. If the foreign genetic material encodes a replicationdefective virus as described in U.S. Ser. No. 08/803,484 “Methods andCompositions for Protective and Therapeutic Genetic Immunization” whichis incorporated herein as if set forth in full, the altered target cellsmay then present viral antigens and also express viral particles andproteins in the lymphoid organs, thereby generating an effectivecellular immune response as well as a humoral immune response.

DETAILED DESCRIPTION OF THE INVENTION

The subject invention most closely concerns methods and compositions forthe delivery of foreign genetic material into cells. It is particularlyuseful to enhance the efficiency of genetic immunization by increasingthe efficiency of gene transfer to certain cells participating in theimmune system, such as antigen presenting cells (APCs). Although thegoal of the inventors is to deliver genetic material into cells, theinventors contemplate that any molecule of suitable size andconfiguration can be delivered into cells using the present invention.Thus, other materials such as drugs or proteins, for example, can bedelivered to targeted cells using the techniques described herein.

The present invention takes advantage of some of the natural pathwaysavailable in animals.

It is known, for example, that specific proteins are imported into cellsby a process called receptor-mediated endocytosis. In this process,specific proteins, or ligands, bind to specific receptors in the plasmamembrane of a cell. The membrane forms a vesicle, or pocket, around theprotein and eventually internalizes the ligand. That is, it imports theprotein into the cell. Afterward the endosome typically delivers thesecomplexes to a lysosome where they are digested into their componentparts, peptides. In cells where MHC expression occurs, peptide-MHCcomplexes accumulate in the lysosome and then reach the surface of thecell in a process called antigen presentation. Receptor-mediatedendocytosis is the means of delivering large molecules such as essentialmetabolites, hormones and growth factors to cells. It is a pathwayexploited by many viruses and toxins to gain entry into cells, and alsoplays a part in the immune response. For example, phagocytic cells havereceptors enabling them to take up antigen-antibody complexes.

Particles complexed with antibodies such as IgG or complements such asC3b, or both, can efficiently enter into cells through receptor-mediatedendocytosis and phagocytosis. Antibodies, or immunoglobulins, havedifferent portions that allow them to perform different functions. Forexample, Immunoglobulin G (IgG) is a Y-shaped molecule with two F_(ab)segments having antigen binding sites and an F_(c) segment whichmediates effector functions. Multivalent antigens can bind to antibodiesand form immune complexes. The size of these immune complexes is afunction of the relative concentration of antigen and antibody.

Endocytosis can be enhanced by a process known as opsonization.Opsonization is a process whereby antibodies coat antigens, therebyproviding a means for other components of the immune system to recognizeand respond to the antigens. Immunoglobulins with the appropriate F_(ab)sites can be used to coat the antigen particles, and subsequently, cellsexpressing the corresponding F_(c) receptors can recognize the F_(c)part of the opsonized antigens and readily endocytose them. Complementssuch as C3b, C4b and C3bi also have opsonization activity. Larger immunecomplexes are more effectively phagocytosed than small ones by cellssuch as B-cells, mononuclear phagocytes, granulocytes, neutrophils anddendritic cells expressing receptors for the F_(c) portions ofimmunoglobulin molecules.

IgG antibodies can be made in animals by injecting the antigen with orwithout adjuvants. Also, antibodies can be cloned and humanized usingmolecular genetic techniques. Other receptors that are commonly locatedon the membranes of immune system cells include, for example,transferrin, mannose and asialoglycoprotein receptors, which couldeasily be used for the transduction of immune system cells. The F_(c)receptor part of the antibody can also be replaced with otherreceptor-binding domains using molecular genetic techniques. For theinventors' present purpose, the F_(c) receptors and corresponding IgGmolecules are convenient, as these serve the further function oftransporting genes to dendritic cells.

Once a molecule or particle is taken up into a cell via endocytosis orphagocytosis, it is contained in a protein-receptor complex called anendosome. Endosomes are intracellular acidic compartments that serve asorting function. Phagosomes, which result from phagocytosis, are large(10×-20×) endosomes. Endosomes then fuse with lysosomes where thematerial is digested to smaller products such as peptides, nucleotidesand sugars. In the present invention, the role of the vector is toprovide the foreign gene to the cell and avoid degradation of the gene.That is, the vector must be able to break the endosome and release thegene into the intracellular fluid, cytosol, or onto the nucleus. Anumber of particles are known to be able to break the endosome afterreceptor-mediated endocytosis, including viral gene delivery particlessuch as adenovirus vectors, retrovirus vectors, pox-virus vectors, andSV-40 virus. Non-viral gene delivery particles include conjugates of DNAwith polylysine, polyethylenimine and its derivatives, liposomes,virosomes and chemicals which increase the pH in the endosome, such aschloroquine.

Target Cells

This invention can be used with any cells capable of receptor-mediatedendocytosis or phagocytosis. The target cells must express a receptorsite which, upon binding with a complementary molecule, can bring thedesired molecule into the endosome or phagosome. For the inventor'spresent purpose, such cells are preferably cells which participate inthe immune response. They include cells which can engage inreceptor-mediated endocytosis and phagocytosis of antigens. Such cellsinclude, for example, B-cells, mononuclear phagocytes, granulocytes anddendritic cells. These cells express receptors for the Fc portion ofimmunoglobulins or complement receptors, or both. Dendritic cells andmacrophages are particularly preferred, because they can efficientlypresent foreign antigens, thereby provoking cellular immune response, orCTL response. The cells can also be targeted through other receptorssuch as the transferrin and mannose receptors.

Dendritic cells reside in the lymphoid tissues, such as the spleen,tonsils and lymph nodes, but they can be found in the blood, epidermis,mucosa, and other peripheral tissues. These cells pick up antigens andmigrate with the antigens to the lymphoid tissues. In the skin,dendritic cells called Langerhans cells can be found in the epidermis.When they endocytose an antigen, they migrate into regional lymph nodes.In the lymph node they are called interdigitating cells, and theypresent the antigen to naive T-cells, provoking the cellular immuneresponse.

To increase the efficiency of gene transfer, the number of availabledendritic cells should be maximized. Choice of location can be a factor.High concentrations of dendritic cells are found, for example, in theskin and on mucosa, such as the mouth, vagina and rectum. Immature DC inthe tissues can efficiently endocytose, therefore they are a good targetof the gene delivery complex which delivers genes with receptor-mediatedendocytosis. However, for efficient expression of MHC molecules andantigen presentation, DC must also be activated. In vitro, immature DCcan be generated from peripheral blood with GM-CSF and IL-4 or from bonemarrow precursors with GM-CFS. Activation of these immature DC can beinduced in vitro and in vivo by bacterial products such aslipolysaccharid and TNF-alpha (Watts C. Nature 338: 724-725, 1997).

Dendritic cells can be attracted to a specific location and activated byan event implicating the immune system such as a cell or tissue injury.For the present purpose, attraction and activation of antigen presentingcells, including dendritic cells, can be mediated by an immune responseunrelated to vaccination or viral infection. An example would be theskin rash that is the result of contact sensitivity to chemicals such asdrugs and toxins, cosmetics and environmental antigens. If a chemicalirritant is swabbed onto the skin, a rash or lesion will usually appear24-48 hours after exposure. The lesion is due to neoantigens created bybinding chemicals to the surface proteins of Langerhans cells.Neoantigens are covalently modified “normal” proteins (e.g.phosphorilated) which are recognized by antibodies. At this site, higherthan normal amounts of dendritic cells may be found, and they are morelikely to be activated, that is, more receptive to immediate endocytosisof an antigen. The choice of irritant depends on its efficacy to attractDC and on its side effects. In another embodiment of the invention, animmune complex-mediated injury can be created. In that case, immunecomplexes with both antigen and antibody components can be used toactivate B cells and the complement cascade with resultant tissueinjury.

Since the cells are targeted through a specific class of receptors, aparticular advantage of this invention is that the gene delivery complexcan be made to target specific cells. If the gene delivery complex ismade with IgG or a polyethylenimine modified with an appropriate starchor sugar, it will be taken up mainly by antigen presenting cells. Thiswould be a great advantage in the development of gene-based vaccines.Targeting other cells expressing, for example, complement receptors ortransferrin receptors is also feasible as described above.

Gene Delivery Complex

The gene delivery complex of the present invention can be used todeliver genes in vitro or in vivo to cells carrying a given receptor.The gene delivery complex is built from two parts: the genetic materialand a delivery particle, and may further comprise a carrier (See FIG.1). In one embodiment, the genetic material is derived from anattenuated HIV virus and the delivery particle is non-viral vector.

In another embodiment, the same or different genetic material may becombined with a viral vector and a carrier. In that instance, thecarrier is preferably an antibody that has a site complementing areceptor present on the surface of the target cell, and an antigenbinding site specific to the desired delivery particle. Such a carrieris specific to both the cell and the delivery particle. For geneticimmunization, human IgG specific to the gene delivery particle isconveniently selected. If there is no antibody commercially available,it can be made with techniques known to one of ordinary skill in theart. If the gene delivery particle is replication-defective HIV-1, humanIgG can be used as a carrier, and is available in large quantities forpassive immunotherapy. The gene delivery particle can be complexed withthe antibody by incubating them together for 5 minutes at roomtemperature. The relative amounts of gene delivery particle and antibodyare determined by whether it is desired to opsonize the gene deliveryparticles.

The Genetic Material

The genetic material, either DNA or RNA, is carried by the deliverycomplex. One or more genes can be encoded on a strand of plasmid DNA, ondouble-stranded DNA or on RNA. Alternatively, the genetic material canbe built into recombinant viruses if they are used as a gene deliveryparticle. If the purpose of the gene transfer is to induce an immuneresponse, then the genetic material must express one or more immunogenicproteins. Transduced cells will subsequently express enough of theimmunogenic proteins (different viral antigens and produce authenticenough viral particles) to provoke a sufficient immune response (e.g.,protect the individual from infection by the wild-type virus).

The choice of the gene delivery particle will be determined by thedisease and the choice of gene(s) to transfer. Where it is desired toconstruct a vaccine for a reverse-transcriptase dependent virus such asIIIV, the DNA preferably encodes at least a substantial portion of areplication-or integration defective virus or the replication- orintegration-defective virus itself. Examples include but are not limitedto integrase negative mutants of a dual-tropic primary isolate such asHIV-1/LW, and derivatives thereof having a deletion in the proteasecleavage site of the gag gene or where the DNA further includes one ormore stop codons in one or more reading frames of the integrase gene.See Methods and Compositions for Protective and Therapeutic GeneticImmunity, U.S. Ser. No. 08/803,484 filed Feb. 20, 1997 and incorporatedby reference as if set forth in full. Where it is desired to construct avaccine for cancer, the immunogen is preferably DNA encoding one or moreoncogens. Other DNA constructs can be DNA encoding replication defectiveHuman Papilloma Virus (causing cervical cancer), replication defectiveHepatitis A, B and C viruses (causing hepatitis and liver cancer), andDNA encoding replication defective animal viruses like Bovine LeukemiaVirus or Feline Immunodeficiency Virus. Choices for a delivery particleincorporating the foreign genetic material can include: (a) replicationdefective HIV or other retrovirus; (b) recombinant adenovirus; (c)plasmid or linear DNA or RNA complexed with PEI or a derivative of PEI;(d) a virosome containing any DNA or RNA; (e) liposome containing DNA orRNA; (f) plasmid DNA-polylysine virus complex; (g) sugar complexed withany DNA or RNA.

The Delivery System

In order to effectively deliver the genes to the cell, the gene deliverysystem must contain the gene or genes to be delivered and also must havethe capacity to break the endosome (or phagosome), rather than bedelivered to a lysosome or be isolated on the outside surface of a celland targeted for destruction. Further, the gene delivery system mustfacilitate incorporation of the foreign genetic material into thegenetic material of the cell.

The gene delivery system can include either a viral or non-viral vector.Viral gene delivery systems include recombinant virus vectors such asadenovirus vectors, retrovirus vectors, pox-virus vectors, mutantviruses (described above) and virosomes. Non-viral gene delivery systemsinclude DNA conjugates with sugar, polylysine, polyethylenimine,polyethylenimine derivatives, and liposomes, together with theirderivatives.

Non-viral gene delivery systems such as those utilizing sugars, sugarderivatives, liposomes, liposome derivatives and polyethylenimine orpolyethylenimine derivatives are preferred. Of these, sugar andpolyethylenimine derivatives adapted to target the mannose receptors ofimmune system cells are most preferred.

Non-viral gene delivery systems offer several advantages over viral genedelivery systems: 1) First, the non-viral vector is not recognized bythe immune system, so no immune response is generated against it. As aresult, it is more likely that individuals treated with the ultimatevaccine will tolerate and develop adequate immune response in cases ofrepeated immunization; 2) non-viral systems are potentially more safethat viral systems because there is no possibility that the system willmutate in an unexpected fashion; 3) non viral systems can be chemicallysynthesized in a large amounts, and are therefore potentially lessexpensive.

The preferred embodiment is based on a cationic polymer,polyethylenimine(PEI). PEI binds to DNA and makes the complex. ThePEI-DNA complex can enter into the endosome of the skin's antigenpresenting cells, Langerhans cells, via asialoglycoproteinreceptor-mediated endocytosis. Then, the PEI component of this complexutilizes endosome buffering and swelling as an escape mechanism to thecytoplasm [Pollard H; Remy J S; Loussouarn G; Demolombe S; Behr J P; JBiol Chem Mar. 27, 1998; 273(13):7507-11]. PEI can also be modified totarget other receptors. For example, a PEI derivative, such as asugar-modified PEI, obtains similar results, except that it is taken upthrough the cells' mannose receptor. Such derivatives can be made in thelaboratory. For example, an isothiocyanantophenyl phenyl mannosederivative can be coupled to PEI 25 kDa, yielding a ligand (or, mannoseresidue of low affinity for the mannose receptor, 1 mM). Anotherpossibility is to use linear PEI 22k Da derivatized mannotenpaoseligand. (These materials were graciously supplied by Dr. Jean-Paul Behr,Laboratoire de Chimie Genetique, Faculte de Pharmacie, CNRS-UMR 7514 74route du Rhin 67401 Illkirch, France)

The mannose receptor is a 175-kDa transmembrane glycoprotein thatspecifically expressed on the surface of macrophages and Langerhanscells. The ectodomain of the mannose receptor has eight carbohydraterecognition domains. The mannose receptor recognizes the patterns ofsugars that adorn a wide array of bacteria, parasites, yeast, fungi, andmannosylated ligands. [Takahashi K; Donovan M J; Rogers R A; Ezekowitz RA, Cell Tissue Res 1998 May; 292(2):311-23]. In contrast to F_(c)receptor, the mannose receptor reconstitutes itself while releasing itscargo [Stahl et al. Cell 1980 19:207]. It thus can internalize ofligands in successive rounds, in a manner similar to the transferrinreceptor, providing a sustained capacity for antigen capture[Goldstein,et al, 1985, Annu Rev Cell Biol. 1:1]. It has been recently discoveredthat mannose-receptor-mediated uptake of antigens results in about 100fold more efficient antigen presentation to T-cells, as compared toantigens internalized via fluid phase [Engering et al. 1997, Eur. J.Immunol. 27:2417-2425]. This enhanced antigen presentation is due tohighly efficient uptake of antigens via the mannose receptor. For thesereasons we believed that targeting the mannose receptor may yield bothspecificity for antigen presenting cells and improved efficiency offunctional uptake of the complex into the endosome.

The Carrier

The carrier of the present invention is the part of the gene deliverycomplex which joins a gene delivery system with a cellular receptor. Inone embodiment the carrier is an immunoglobulin G (IgG). IgG is aY-shaped molecule with two F_(ab) segments having antigen binding sitesand an F_(c) segment which binds to the cellular receptor calledF_(c)-receptor. Immune system cells such as B-cells, mononuclearphagocytes, granulocytes and dendritic cells have F_(c) receptors. WhenIgG is used as a carrier, it targets specifically cells having F_(c)receptors.

To change the carrier specificity to target other cells, the F_(c) partof the antibody can be replaced by other receptor binding domains, suchas complement, sugar, or transferrin.

Where the carrier is an antibody large complexes are formed, the carrierand gene delivery system are preferably combined in equal proportions.Where it is desirable to opsonize the particle, the amount of thecarrier greatly exceeds the amount of the gene delivery particles. Bothendocytosis and phagocytosis are enhanced in the case of large complexesand opsonized particles. The gene delivery complex is preferablyopsonized with the carrier. Where an opsonized gene delivery complexmade of an antibody complexed with a delivery particle incorporatingforeign genetic material is administered to an individual, cellularimmune response will be maximized over the humoral immune response. Thedendritic cells will be activated by the opsonized complexes, andendocytosis will be more efficient. Also, the multiple antibodies willblock the antigenic determinants (epitopes) of the delivery particle.Therefore, no direct antibody response to the delivery particle would beexpected. Also, some antibody complexed antigens will bind to the F_(c)receptor site of B cells, further inhibiting their antibody response.However, cellular immunity would be stimulated because the complex wouldbe endocytosed or phagocytosed by various kinds of antigen presentingcells, including dendritic cells and macrophages.

In a preferred embodiment, the carrier is covalently joined to the nonviral gene delivery system. PEI can be chemically modified with sugars(e.g., mannose, glucose, galactose, etc.). The carrier in this case isthe sugar ligand, which is recognized by the mannose receptor. To changethe carrier's specificity, sugar can be replaced by otherreceptor-binding domains.

In vivo Gene Delivery

The gene delivery complex can be injected directly into the blood, skin,or other place where cells corresponding to the carrier bindingspecificity are located. The complex can be applied on the skin ormucosa surfaces directly. In that event, it is preferable that theLangerhans' cells are activated on the surface. Activation may beachieved by receptor stimulation (e.g., mannose receptor), toxinactivation (cholera toxin), a tissue or cell injury such asinflammation, and may be the consequence of another antigenicstimulation.

The complex can be infused using a pediatric feeding tube orally,vaginally or rectally in the case of human or animal adults or neonates.Neonates may respond better to oral administration than adults.Alternatively, the gene delivery complex may be packaged in asuppository and inserted in the vagina or rectum.

Where a viral delivery particle is used, the delivery particle can beinjected directly into the muscle or skin, in the presence or absence ofadjuvants, of the subject on two separate occasions for high titer ofantibody production in vivo. The first injection will result primarilyin a humoral immune response. That is, the capability to produce largenumbers of antibodies will result. Where a concentration of IgGantibodies sufficient to opsonize the delivery particles is available,(it can be measured or assessed by experience) then the deliveryparticle can be administered a second time as described in 1-3 above.The site of the second administration must be chosen carefully to ensurethat cells are present which can phagocytose or endocytose the opsonizedantigens.

Treatment of Active Infection

The vaccine of the present invention might also be used as a method oftreating active HIV infection. HIV replicates abundantly, mutatesrapidly, and damages the immune system. Both the rate of replication andthe rate of mutation outpace the immune system's ability to respond.This means that, while the immune system is capable of mounting aneffective response to a given type of HIV particle, enough new variantsof the particle are produced to stay ahead of the immune system. Ifreplication of the wild-type virus can be suppressed either before theimmune system is substantially damaged or long enough to allow theimmune system to recover, the vaccine of the present invention can beused to strengthen the immune system's ability to recognize the newvariants of the virus, thereby providing a means of controlling viralreplication in individuals that have already been infected.

Drug combinations that are effective to at least temporarily inhibit HIVreplication are known. The inventors have shown that drug combinationsincluding hydroxyurea, one or more reverse transcriptase inhibitors and,optionally, one or more protease inhibitors are particularly effective,and, for some patients, allow the possibility of stopping drug treatmentfor extended periods of time. See U.S. Ser. No. 09/056,691, filed Apr.8, 1998, U.S. Pat. No. 5,977,086, “Method of Inhibiting HIV by CombinedUse of Hydroxyurea, a Nucleoside Analog, and a Protease Inhibitor, U.S.Ser. No. 09/048,886, filed Mar. 26, 1998, U.S. Pat. No. 6,251,874 Methodof Inhibiting HIV using Hydroxyurea and Reverse Transcriptase Inhibitorin vitro and U.S. Ser. No. 09/048,576, now abandoned filed Mar. 26,1998, Method of Rendering a HIV Population Replication Incompetent invivo, all of which are incorporated herein by reference as if set forthin full. The present invention includes the treatment of a patient withactive HIV infection with an appropriate drug combination until theviral load in the blood has reached a suitably low level, less thanabout 50,000 copies per ml, preferably less than 10,000 copies per ml,more preferably less than 200-500 copies per ml. The patient is thenvaccinated using the present invention while the drug combinationsuppresses replication of the wild-type virus.

The following Examples are presented for the purpose of illustrating thepractice of the present invention. They do not limit the invention, orthe claims which follow.

EXAMPLES

1. Expression of Plasmid DNA Encoding a Replication Defective HIV inCultured DC

There are several sources of DC. DC can be isolated from bone marrowCD34+ hematopoietic progenitor cells. Bone marrow mononuclear cells willbe separated by Ficoll-Hypaque gradient centrifugation. These cells willbe positively selected with human CD34 antibodies conjugated magneticbeads (Dynal Detachabeads) and CD34+ cells will be displaced frommagnetic beads using high affinity polyclonal antibody against CD34monoclonal antibody. These cells can differentiate to DC when they arecultured with stem cell factor, GM-CSF and TNF-alpha [Canque, B., M.Rosenzwajg, et al. (1996). “The effect of in vitro humanimmunodeficiency virus infection on dendritic-cell differentiation andfunction.” Blood 88(11): 4215-28.]

Monocyte-derived DC were generated from peripheral blood mononuclearcells in the presence of GM-CSF and IL-4. [Bender, A., M. Sapp, et al.(1996). “Improved methods for the generation of dendritic cells fromnonproliferating progenitors in human blood.” J Immunol Methods 196(2):121-35.] On day 4, cells were transfected with lipofectamine complexedwith plasmid DNA encoding HIV-1/LWint- (an integration and replicationdefective HIV described in U.S. Ser. No. 08/803,484). Lipofectamine, acommercially available cationic liposome useful as a transfectionreagent (available from Gibco BRL Life Technology, PM. Gaithersburg,Md., US) 48 hours later cells were washed and analyzed. The purity ofthe DC, characterized by Fluorescence Activated Cell Sorter (FACS)measuring surface markers (FACS) was 90.6%. DC cell types were found tobe CD3-, CD19-, CD56-, CD14- and HLA DR+ using FACS analysis. Theexpression of HIV-1 Gag and Env and Tat proteins was also measured byFACS in permeabilized cells. The level of non-specific binding ofisotope control Ig was the same in the control transduced and specificplasmid transduced. We found in three independent experiments that25-37% of HIV-1/LWint-tranduced DC expressed Env, Gag and Tat proteins.That is, 25-37% of the cells in the transduced samples expressed HIVproteins. Transduced and control cell samples were also double-stainedwith p24 and B7-2 antibodies to demonstrate that DC and not macrophageswere expressing the antigen. These results were surprisingly good,because using the same methods with another plasmid DNA (CMV-drivenhemagglutinin of influenza virus gene) only 5-8% of the transfectedcells expressed proteins. These results demonstrated that defective HIVcan be efficiently expressed by transduced DC.

2. DNA Encoding Replication Defective Viruses are More EfficientAntigens than DNA Encoding One or More Proteins

In an independent experiment we compared the expression of two differentHIV plasmids in DC: HIV-1/LWint- and LTR-tat. Both constructs are drivenby the same promoter: HIV-1-LTR and the expression of both constructsdepends on the transactivation of Tat. Transfection was performed asdescribed in example 1 and 48 hours later expression of the Tat proteinwas analyzed by FACS. We found that 32% of HIV-1/LWint- plasmidtransfected DC expressed Tat protein. In contrast only 10% of theLTR-tat transfected DC expressed the same Tat protein. This result wassuprising, because in this comparative study we were expected the sameefficiency with the two different constructs. Replication defectiveviruses definitly have the capability to form viral particle, which canbe released from the cell. Since antigen presentation depends on geneexpression in DC, this experiment clearly demonstrate that DNA encodingdefective viruses are more efficient antigens than DNA encoding one ormore proteins.

3. Transduced, Cultured DC Activate Naive CTL in vitro

After transduction, DC were cultured with autologous T-cells at a ratioof 1:10. After 7 days these T-cells were used as an effector to lyse(kill) a monocyte/macrophage cell population from the same donor thathad been pulsed with p55, an HIV protein conveniently used as a label.This CTL activity was measured using Cr-relase assay. As FIG. 5demonstrates, CTL induced by transduced DC lysed the target cellsspecifically and effectively. Because generation of CTL in vitro is moredifficult than in vivo, these experiments show that cells which havebeen subjected to genetic immunization can activate naive CTL so thatthey will effectively lyse in vivo infected cells. Furthermore, theexperiment further demonstrates that DNA encoding a defective virus notonly express efficiently HIV genes but also can generate an effectiveimmune response.

4. Ex Vivo Genetic Immunization with Transduced DC

To prove the in vivo efficacy of genetic immunization in monkeys,dendritic cells were generated from 40 ml peripheral blood of pigtailmacaques. Cells were transfected with LW/int- plasmid usingpolyethylenimine as described in Example 5. The transfected DC werewashed and injected into juvenile pigtail macaques 36-48 hours aftertransfection. One part of the transfected DC was injected subcutaneouslyand one part was injected intravenously. After 4 weeks and only oneimmunization attempt, one monkey already showed CTL response (FIG. 6),which suggests that the in vitro result can be reproduced in vivo inanimals. In contrast, the HIV subunit vaccines presently approved forPhase III human clinical trials have not been shown to generate any CTLresponse, even after multiple immunization attempts.

5. Polyethyleneimine-mediated Gene Transfer into Cultured DendriticCells

Dendritic cells were transduced with plasmid encoding HIV-1/LWint- as inExample 1, except that polyethyleneimine (PEI kindly supplied by Dr.Behr) was used instead of lipofectamine. The cells were tested as inExample 1, and more than 60% of the dendritic cells transduced usingpolyethyleneimine expressed HIV-1 proteins in contrast to the 25-37% ofcells transduced using lipofectamine. Since up to date lipofection wasthe best gene transfer method to introduce plasmid DNA into DC, thisexperiment demonstrates that PEI is the most efficient non viral genedelivery system to transfer genes into DC. However, both PEI andlipofectamine exhibited significant toxicity to dendritic cells, asmeasured by tripan blue staining.

6. Specifically Targeting DC via Mannose Receptor

Immature DC were generated as described above in Example 1 andtransfected with DNA encoding a green fluorescent protein (GFP). We usedthis DNA as a marker for gene expression, because cells expressing thegreen fluorescent protein light up green after the fluorescentstimulation. Therefore, transfected cells can be visualized byfluorescent microscopy and flow cytometry (FACS).

PEI modified with different sugars was chosen to target the mannosereceptor on the surface of dendritic cells because the mannose receptorrecognizes all the patterns of sugars on the surface of bacteria,parasites, yeast and fungi. DNA was complexed with PEI and withdifferent sugar-bearing polyethylenimines (available on a custom orderbasis from Dr. Jean-Paul Behr, Laboratoire de Chimie Genetique, Facultede Pharmacie, CNRS-UMR 7514 74 route du Rhin 67401 Illkirch, France). 2microgram DNA was incubated with different derivates of PEI in 150 mMNaCl (10:1 N:P ratio) at room temperature for about 5 minutes. Than DCwere transduced with the complexes for 6 hours washed, and greenfluorescent cells were analyzed after 48 hours. We found that the mosteffective PEI-sugar modification is the PEI-mannose (Table 1).

TABLE 1 Different Complexes for in vitro Transduction of Dendritic cells% of cells expressing green Experiment fluorescent protein 1. Control  43. PEI-mannose-DNA 43 4. PEI-galactose-DNA 23 5. PEI-glucose-DNA 19

7. In Vitro PEI-Mannose-mediated Gene Transfer into Cultured DendriticCells

The mannose-bearing polyethylenimine (PEI-man) is anisothiocyanantophenyl phenyl mannose derivative, coupled to PEI 25 kDa,yielding a ligand (or, mannose residue of low affinity for the mannosereceptor, 1 mM). It has been previously demonstrated that entry via theasialoglycoprotein receptor (used by PEI) requires the complex to becharged. That is, more PEI than DNA must be used. When the complex isneutralized, that is, the PEI is neutralized by the DNA, the complexcannot enter via the asialoglycoprotein receptor. [Zanta M A; Boussif O;Adib A; Behr J P. Bioconjug Chem November-December 1997; 8(6):839-44]:These investigators developed a hepatocyte-directed complex; it includesseveral key features thought to favoring vivo gene delivery to theliver: 1) electrostatically neutral particles which avoid nonspecificbinding to other cells, and 2) to avoid asialoglycoproteinreceptor-mediated endocytosis. This system was based on a 5%galactose-bearing polyethylenimine (PEI-gal) polymer which is condensedwith plasmid DNA to neutrality.

We found that, with PEI-man-DNA complexes, less DNA is required toneutralize PEI-man compared to PEI. Gel electrophoresis experimentsusing different N:P ratio of PEI-DNA complexes demonstrated that 5:1(N:P) of PEI-man:DNA complex has neutral charge, in contrast to 3:1(N:P) PEI-DNA complex. {The neutralization of PEI with the DNA dependson the N(nitrogen):P(phosphate) ratio; one microgram DNA=3×109 molar Pand 1 mM PEI=109 molar N/microliter. This means for example that 10:1ratio is the mixture of 3 microliter 10 mM PEI and 1 microgram DNA.}

Human DC were isolated as described above. Purity of DC, characterizedby FACS, was over 99%. By measuring cell viability with tripan-bluestaining, we found that PEI-man was much less toxic than PEI. We alsofound that both PEI and PEI-man are able to introduce DNA into DC,however PEI-mannose is more efficient. At 5:1 (N:P) ratio PEI-man-DNAcomplex is neutral, therefore the complex is only able to enter into DCvia the mannose receptor. Under these conditions, 30% of the DC wereexpressing the green fluorescent protein. In contrast, 5:1 (N:P) ratioPEI-DNA complex is charged and the complex enters via asialoglycoproteinreceptor. Under this condition, 14% of the DC expressed the greenfluorescent protein (FIG. 4).

8. In Vivo Gene Delivery to Skin Langerhans Cells

In the skin, the only cells which can endocytose mannosylated ligandsare the Langerhans cells. Therefore, the main question was whether thePEI-(man) complex can penetrate into the skin and transfect Langerhanscells in vivo. PEI-(man) was complexed with plasmid DNA encoding a greenfluorescent protein (GFP). Experiments with different gene deliverycomplexes were performed as described in FIG. 3. The complex contained50 microgram DNA and 8.25 microliter 100 mM PEI-man in a 5-10% glucosesolution (optimum=8%). BALB/c mice were anaesthetized, and the backs ofthe mice were shaved. 0.1 ml of the complexes were applied on the skinfor one hour or subcutaneously as indicated in the Table 2. Mice weresacrificed 6 hours after immunization and skin samples were placed inDMEM media supplemented with 10% fetal calf serum and antibiotics. Underthese conditions cells, including Langerhans cells migrate out from theskin. One day later the migrating cells were collected and analyzed byflow cytometry (FACS), because this analysis can recognize cellsexpressing the green fluorescent protein. In our analysis only the largeand dense cell population was analyzed, because both dendritic cells andLangerhans cells are known to be large, dense cells.

TABLE 2 In vivo transduction of skin Langerhans cells % of cellsexpressing green Experiment fluorescent protein 1. Control 0.84 2.Subcutaneous PEI-DNA 0.20 3. Subcutaneous DNA 1.74 4. TranscutaneousPEI-DNA 6.52 5. Transcutaneous DNA 29.10 6. Transcutaneous PEI-man-DNA22.99

These experiments demonstrate that 1) transcutaneous gene deliveryresults in more efficient gene transfer into Langerhans cells thansubcutaneous delivery (compare experiment 2 & 4 or 3 & 5). This isimportant, because one of the best present vaccination technologies usessubcutaneous injection. 2) Entry via mannose receptors is more efficientto transfect in vivo Langerhans cells than entry via asialoglycoproteinreceptor (compare experiment 4 & 6). These in vivo experiments confirmour in vitro experiment (see FIG. 4). Therefore, the sugar modified genedelivery system is preferred to transduce antigen presenting cells.

9. Transduced Langerhans Cells Migrate to the Lymph Nodes

BALB/c mice were prepared as in Example 8, and 0.1 ml samples of thePEI-man-DNA complex were applied on the skin for one hour. 2 days laterthe animals were sacrificed and lymph nodes (LN) were removed. AuxiliaryLN were investigated because they are the draining LN of the back, andmigrating Langerhans cells might be found there. The LN were frozen,sliced and examined under fluorescent microscope. The LN of experimentalmice were compared with the LN of a control mouse. FIG. 7 demonstratesgreen fluorescent cells in the LN of the experimental animal. We wereable to detect about 15 green fluorescent cells in the samples from theexperimental LN and none in the control LN. These results demonstratethat the complex entered into cells located in the skin, and the cellswere able to migrate into the LN and express the green fluorescentprotein. The morphology of these green cells resembles DC morphology:these are big cells and the localization of the green fluorescence showsa “bumpy” pattern, which is characteristic to DC. (Other cells, e.g. 293cells show a diffused green pattern in the cytoplasm.) In addition, theonly cell type is able to pick up antigens and migrate to the LN is theLangerhans cell. These cells are the only cells in the skin to bear themannose receptor in order to take up the complex and after activation isknown that they are migrating in the draining LN.

These experiments show that PEI-(Man)-DNA complexes are able topenetrate in the skin, and deliver the DNA into Langerhans cells. TheLangerhans cells were activated and migrated into the draining LN andexpressed genes encoded by the DNA construct in the LN. It is known thatcultured DC reinjected to the body migrate in the LN and generateefficient immune response. This invention demonstrates that in vitroisolation of DC is not required to transfer genes into Langerhans cells,or for gene expression in the lymphoid organs. We have also demonstratedthat expression of a replication defective virus in DC results inefficient induction of a CTL response in vitro and in vivo (see above).Therefore, we have shown that transcutaneous gene delivery withcomplexes (like PEI-man-DNA) can be utilized to generate immuneresponses against proteins encoded in the DNA.

10. Sugar-DNA Complexes to Transduce Skin Langerhans Cells

Experimental results depicted in Table 2 provided evidence that asugar-DNA complex, in the absence of PEI-man, can transduce Langerhanscells in vivo. Sugar complexed DNA in the absence of PEI is moreefficient for use in both subcutaneous and transcutaneous methods thanDNA complexed with PEI (see Table 2, experiments 3 & 5). This is a verysurprising result. It shows that sugars (e.g. 8% glucose in theseexperiments) can also complex DNA and deliver the DNA to the Langerhanscells via the mannose receptor. Importantly, the most efficient genedelivery in vivo to the Langerhans cells was the sugar complexed DNAused in the transcutaneous way.

We expect that immunization with the sugar-DNA complex would also resultin migration of the Langerhans cells to the draining lymph node. Thereason is that the same mechanism is utilized for entry to Langerhanscells: mannose-receptor mediated uptake. The advantage of using sugarsas adjuvants in the presently used vaccination technologies is thathigher percentages of Langerhans cells will be involved in generation ofimmune response. This is expected to significantly increase the efficacyof present vaccination strategies. For example, mixing vaccines withsugar for subcutaneous, intradermal and intramuscular injections of DNAand protein antigens.

11. Implications of Transcutaneous Immunization

This technology would revolutionize the immunization methods because noneedles are required. The described transcutaneous immunization is avery simple technology which could be used for inexpensive vaccinationin both the developed and developing world.

The simplicity of the methodology and the fact that the antigenpresenting cells are transduced efficiently enables us to use any DNAsequence able to generate an immunogenic protein. Thereby, the broadestspectrum of diseases can become target of immunization, e.g. infectiousdiseases and cancer.

The only way to eradicate infectious diseases, like HIV infection,hepatitis, malaria, is a simple inexpensive vaccination.

Vaccines without needle sticks will be expecially welcome to the parentsof small children.

Use of the present invention potentially allows the development of safervaccines. The few negative reactions to classical vaccines are sometimesdue to an allergic reaction to byproducts of the vaccine manufacturingprocess. Where such additional materials (egg albumin, for example) canbe eliminated, the rate of negative reactions can be concomitantlyreduced.

It can be used for immunization for the treatment of diseases with orwithout combination chemotherapy.

What is claimed is:
 1. A gene delivery complex comprising DNA andmannosylated polyethylenimine, wherein said DNA comprises a nucleic acidsequence encoding at least one immunogenic protein operatively linked toa promoter.
 2. The gene delivery complex of claim 1, wherein theimmunogenic protein is derived from a reverse transcriptase-dependentvirus.
 3. The gene delivery complex of claim 2, wherein the reversetranscriptase-dependent virus is a human immunodeficiency virus.
 4. Thegene delivery complex of claim 3, wherein the human immunodeficiencyvirus is replication-defective.
 5. The gene delivery complex of claim 3,wherein the human immunodeficiency virus is integration-defective.
 6. Acomposition for delivering DNA into antigen presenting cells comprisinga glucose solution, DNA, and mannosylated polyethylenimine, wherein saidDNA comprises a nucleic acid sequence encoding at least one proteinoperatively linked to a promoter.
 7. The composition of claim 6, whereinthe mannosylated polyethylenimine is an isothiocyanantophenyl phenylmannose derivative coupled to PEI 25 kDA.
 8. The composition of claim 6,wherein the protein is an immunogenic protein.
 9. The composition ofclaim 6, wherein the immunogenic protein is derived from a reversetranscriptase-dependent virus.
 10. The composition of claim 6, whereinthe protein is derived from a virus.
 11. The composition of claim 10,wherein the virus is a replication- or integration-defective, reversetranscriptase-dependent virus.
 12. The composition of claim 6, whereinthe protein is an immunogenic protein from a human immunodeficiencyvirus.
 13. The composition of claim 12, wherein the humanimmunodeficiency virus is an integrase-defective human immunodeficiencyvirus.
 14. The composition of claim 1 or 6, wherein the DNA is aplasmid.